Exocyclic DNA adducts and oxidative stress parameters: useful tools for biomonitoring exposure to aldehydes in smokers.

CONTEXT: Exocyclic DNA adducts have been shown to be potential biomarkers of cancer risk related to oxidative stress and exposure to aldehydes in smokers. In fact, aldehydes potentially arise from tobacco combustion directly and endogenously through lipid peroxidation. OBJECTIVE: This study aims to investigate the relationship between a profile of nine aldehydes-induced DNA adducts and antioxidant activities, in order to evaluate new biomarkers of systemic exposure to aldehydes. METHODS: Using our previously published UPLC-MS/MS method, adducts levels were quantified in the blood DNA of 34 active smokers. The levels of antioxidant vitamins (A, C and E), coenzyme Q10, ß-carotene, superoxide dismutase (SOD) and autoantibodies against oxidized low-density lipoprotein were measured. RESULTS: Adducts induced by tobacco smoking-related aldehydes were quantified at levels reflecting an oxidative production from lipid peroxidation. A significant correlation between SOD and crotonaldehyde-induced adducts (p = 0.0251) was also observed. ß-Carotene was negatively correlated with the adducts of formaldehyde (p = 0.0351) and acetaldehyde (p = 0.0413). Vitamin C tended to inversely correlate with acetaldehyde-induced adducts (p = 0.0584). CONCLUSION: These results are promising, and the study is now being conducted on a larger cohort with the aim of evaluating the impact of smoking cessation programs on the evolution of adducts profile and antioxidants activities.

Mesalazine initiates an anti-oncogenic ß-catenin / MUCDHL negative feed-back loop in colon cancer cells by cell-specific mechanisms.

Chronic inflammation associated with intestinal architecture and barrier disruption puts patients with inflammatory bowel disease (IBD) at increased risk of developing colorectal cancer (CRC). Widely used to reduce flares of intestinal inflammation, 5-aminosalicylic acid derivatives (5-ASAs) such as mesalazine appear to also exert more direct mucosal healing and chemopreventive activities against CRC. The mechanisms underlying these activities are poorly understood and may involve the up-regulation of the cadherin-related gene MUCDHL (CDHR5). This atypical cadherin is emerging as a new actor of intestinal homeostasis and opposes colon tumorigenesis. Here, we showed that mesalazine increase mRNA levels of MUCDHL and of other genes involved in the intestinal barrier function in most intestinal cell lines. In addition, using gain / loss of function experiments (agonists, plasmid or siRNAs transfections), luciferase reporter genes and chromatin immunoprecipitation, we thoroughly investigated the molecular mechanisms triggered by mesalazine that lead to the up-regulation of MUCDHL expression. We found that basal transcription of MUCDHL in different CRC cell lines is regulated positively by CDX2 and negatively by ß-catenin through a negative feed-back loop. However, mesalazine-stimulation of MUCDHL transcription is controlled by cell-specific mechanisms, involving either enhanced activation of CDX2 and PPAR-γ or repression of the ß-catenin inhibitory effect. This work highlights the importance of the cellular and molecular context in the activity of mesalazine and suggests that its efficacy against CRC depends on the genetic alterations of transformed cells.

Perception of pharmacy students toward opioid-related disorders and roles of community pharmacists: A French nationwide cross-sectional study.

Background: Community pharmacists are among the frontline health professionals who manage patients with an opioid-related disorder (ORD). Pharmacists frequently have a negative attitude toward these patients, which could have a negative impact on their management. However, education on ORD may improve the attitude of future healthcare professionals. This cross-sectional study aimed to assess French pharmacy students' perceptions of ORD. Methods: This online survey was performed by emails sent to French pharmacy schools (between January 14, 2019 and May 31, 2019). The primary outcome was the perception (visual analogic scale) of ORD as a disease, the roles of community pharmacies (delivery of opioid agonist therapy-OAT and harm reduction kits), and the efficacy of OAT. The secondary outcomes assessed professional experience, university experience of and education on ORD, and the individual characteristics of students. Results: Among the 1,994 students included, 76.3% perceived ORD as a disease and felt that it was normal for pharmacists to deliver OAT (78.9%) and harm reduction kits (74.6%). However, only 46.9% perceived OAT as being effective. Multivariable analyses showed that females had a more positive perception in recognizing ORD as a disease. The progression through university years increased the positive perception of ORD as a disease and the delivery of OAT and harm reduction kits by pharmacists. Education on substance-related disorders had no impact on any scores. Students who had already delivered OAT had a negative perception of their efficacy. The students who had already performed pharmacy jobs or traineeships had a negative perception of harm reduction kit delivery. Conclusion: Education on substance-related disorders had no impact on students' perceptions. It seemed that the maturity acquired through university years had a stronger impact on the students' perceptions of ORD. Efforts must be made to improve our teaching methods and reinforce the confidence of students in the roles of community pharmacists.

Genotoxicity of aldehyde mixtures: profile of exocyclic DNA-adducts as a biomarker of exposure to tobacco smoke.

Electrophilic compounds present in humans, originating from endogenous processes or pollutant exposures, pose a risk to health though their reaction with nucleophilic sites in protein and DNA. Among this chemical class, aldehydes are mainly present in indoor air and they can also be produced by endogenous lipid peroxidation arising from oxidative stress. Known to be very reactive, aldehydes have the ability to form exocyclic adducts to DNA that, for the most if not repaired correctly, are mutagenic and by consequence potential agents involved in carcinogenesis. The aim of this work was to establish profiles of exocyclic DNA adducts induced by aldehyde mixtures, which could ultimately be considered as a genotoxic marker of endogenous and environmental aldehyde exposure. Adducts were quantified by an accurate, sensitive and validated ultra high performance liquid chromatography-electrospray ionization analytical method coupled to mass spectrometry in the tandem mode (UHPLC-ESI-MS/MS). We simultaneously measured nine exocyclic DNA adducts generated during the exposure in vitro of calf thymus DNA to different concentrations of each aldehyde along, as well as, to an equimolar mixture of these aldehydes. This approach has enabled us to establish dose-response relationships that allowed displaying the specific reactivity of aldehydes towards corresponding adducts formation. Profiles of these adducts determined in DNA of current smokers and non-smokers blood samples supported these findings. These first results are encouraging to explore genotoxicity induced by aldehyde mixtures and can furthermore be used as future reference for adductomic approaches.

A validated UHPLC-MS/MS method for simultaneous quantification of 9 exocyclic DNA adducts induced by 8 aldehydes.

Human exposure to aldehydes is implicated in several diseases including cancer. These strong electrophilic compounds can react with nucleophilic sites in DNA to form reversible and irreversible modifications. These modifications, if not repaired, can contribute to pathogenesis. The aim of our study was to provide a mass spectrometry (MS)-based profiling method for identifying potential biomarkers of aldehydes exposure. We have developed and validated a highly sensitive method using ultra high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) for the simultaneous quantitation of 9 exocyclic DNA adducts derived from 8 main exogenous and endogenous aldehydes, namely formaldehyde, acetaldehyde, acrolein, crotonaldehyde, malondialdehyde, 4-hydroxy-2-nonenal, glyoxal and methylglyoxal. Finally, we applied the established method to quantify adducts in genomic DNA isolated from the blood of a smoker and a non-smoker blood samples in order to demonstrate its applicability.

Synthesis and biological evaluation of bile carboxamide derivatives with pro-apoptotic effect on human colon adenocarcinoma cell lines.

We previously reported that the cinnamylpiperazinyl group in the side chain of the chenodeoxycholic acid showed apoptosis-inducing activity on multiple myeloma cancer cell line KMS-11. In the present study, we synthesized and tested the pro-apoptotic potency of fifteen new piperazinyl bile carboxamide derived from cholic, ursodeoxycholic, chenodeoxycholic, deoxycholic and lithocholic acids on human colon adenocarcinoma cell lines DLD-1, HCT-116, and HT-29. Cell viability was first measured using XTT assay. The most of the synthetic bile carboxamide derivatives decreased significantly cell viability in a dose-dependent manner. HCT-116 and DLD-1 cell lines were more sensitive than HT-29 to tested compounds. 9c, 9d showed the best in vitro results in term of solubility and dose-response effect on the three colon adenocarcinoma cell lines. Additionally, flow cytometric and Western-blotting analysis showed that 9c induced pro-apoptosis in DLD-1 and HCT-116 whereas 9d did not. We conclude that the benzyl group improved anti-proliferative activity and that the α-hydroxyl group was found to be more beneficial at the 7-position in steroid skeleton.

Benzo[a]pyrene, aflatoxine B1 and acetaldehyde mutational patterns in TP53 gene using a functional assay: relevance to human cancer aetiology.

Mutations in the TP53 gene are the most common alterations in human tumours. TP53 mutational patterns have sometimes been linked to carcinogen exposure. In hepatocellular carcinoma, a specific G>T transversion on codon 249 is classically described as a fingerprint of aflatoxin B(1) exposure. Likewise G>T transversions in codons 157 and 158 have been related to tobacco exposure in human lung cancers. However, controversies remain about the interpretation of TP53 mutational pattern in tumours as the fingerprint of genotoxin exposure. By using a functional assay, the Functional Analysis of Separated Alleles in Yeast (FASAY), the present study depicts the mutational pattern of TP53 in normal human fibroblasts after in vitro exposure to well-known carcinogens: benzo[a]pyrene, aflatoxin B(1) and acetaldehyde. These in vitro patterns of mutations were then compared to those found in human tumours by using the IARC database of TP53 mutations. The results show that the TP53 mutational patterns found in human tumours can be only partly ascribed to genotoxin exposure. A complex interplay between the functional impact of the mutations on p53 phenotype and the cancer natural history may affect these patterns. However, our results strongly support that genotoxins exposure plays a major role in the aetiology of the considered cancers.

8-oxo-7,8-dihydro-2'-deoxyguanosine as a biomarker of oxidative damage in oesophageal cancer patients: lack of association with antioxidant vitamins and polymorphism of hOGG1 and GST.

BACKGROUND: The present report was designed to investigate the origins of elevated oxidative stress measured in cancer patients in our previous work related to a case-control study (17 cases, 43 controls) on oesophageal cancers. The aim was to characterize the relationship between the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), antioxidant vitamins and genetic susceptibility. METHODS: 8-oxodG was analysed in peripheral blood mononuclear cells (PBMCs) by High Performance Liquid Chromatography with Electrochemical Detection (HPLC-ED). Analysis of gene polymorphisms in GSTM1 and GSTT1 was performed by multiplex PCR and in GSTP1 and hOGG1 by a PCR-RFLP method. Reversed-phase HPLC with UV detection at 294 nm was used to measure vitamins A and E in serum from the same blood samples. RESULTS: We observed that in our combined population (cases and control, n = 60), there was no statistically significant correlation between the levels of 8-oxodG and (i) the serum concentration of antioxidant vitamins, vitamin A (P = 0.290) or vitamin E (P = 0.813), or (ii) the incidence of the Ser326Cys polymorphic variant (P = 0.637) of the hOGG1 gene. Also, the levels of 8-oxodG were not significantly associated with polymorphisms in metabolite-detoxifying genes, such as GSTs, except for the positive correlation with Val/Val GST P1 allele (P < 0.0001). CONCLUSIONS: The weakness of our cohort size notwithstanding, vitamins levels in serum and genetic polymorphisms in the hOGG1 or GST genes do not appear to be important modulators of 8-oxodG levels.

Expression of p53 family members and CD44 in oral squamous cell carcinoma (OSCC) in relation to tumorigenesis.

Oral squamous cell carcinomas (OSCCs) are described as the result of a multistep tumorigenesis process. In order to develop useful diagnosis of pre-malignant lesions, expression of p53 family members and the cancer stem cell (CSCs) marker, CD44v6, were studied in histologically normal oral epithelium, precancerous lesions and succeeding invasive OSCCs. p53 was expressed focally in normal epithelium adjacent to tumors, while expression was high in intra-epithelial neoplasia and moderate in OSCC. p63 nuclear staining was important in basal and suprabasal layers of histologically normal oral mucosa and in immature compartments of premalignant lesions and cancer. In epithelium without neoplasia, intense p73 staining was observed in the basal layer, while focal expression was present in suprabasal layers. Most immature dysplastic areas showed either high or moderate staining, whereas those in OSCCs expressed low and moderate p73 level expression. CD44v6 was only expressed in poorly differentiated areas of epithelium, altered or not. p53, p63 and p73 positive stainings were statistically related in intra-epithelial neoplasia to tumours. Analysis of TP53 mutations in 17% of tumours principally revealed G>A and A>G transitions. No relation was observed between this mutational profile and different immunostainings. In conclusion, our results support that immunostaining of p53 family members might be helpful in diagnosis and monitoring of high-risk pre-malignant lesions of oral epithelium. The combination of staining patterns of p63, p73alpha and CD44v6 enabled us to isolate phenotypic undifferentiated or transient amplifying areas, reflecting the immaturity of the tumour cell lineage. While CD44v6 expression is an interesting marker of such epithelial cells, it is not specific enough to be useful alone and other phenotypic markers are needed.

Aflatoxin B1-induced TP53 mutational pattern in normal human cells using the FASAY (Functional Analysis of Separated Alleles in Yeast).

Mutations in the TP53 gene are the most common alterations in human tumours. In hepatocellular carcinoma (HCC) related to exposure to aflatoxin B1, a specific G>T transversion in codon 249 is classically described as a hot spot. However, AFB1 is suspected to be a potent carcinogen in tissues other than the liver. By using the FASAY functional assay in yeast, the present study aimed at depicting the mutational pattern of TP53 in normal human fibroblasts after in vitro exposure to AFB1. Molecular analysis of mutants revealed that codon 245 was the main hot spot, whereas no mutations were found in codon 249. The locations of mutations within GG and GC/CG sequences are well in accordance with AFB1-adduct location data. In our assay with normal human fibroblasts, AFB1 mainly induced G>A transitions, followed by G>T and A>G mutations. This suggests that G>T transversions at codon 249 were likely the result of a selection bias in human HCC rather than a true fingerprint of AFB1 adducts. Indeed, a comparison of the mutation pattern with that found in human HCC excluding codon 249 reveals that the two spectra are quite similar. Furthermore, the similarity between our in vitro spectrum with that identified in AFB1-induced lung tumours in mice suggests that AFB1 may be a potent lung carcinogen in humans.

Acetaldehyde-induced mutational pattern in the tumour suppressor gene TP53 analysed by use of a functional assay, the FASAY (functional analysis of separated alleles in yeast).

Chronic alcohol consumption is a major risk factor for upper aero-digestive tract cancers, including cancer of the esophagus. Whereas alcohol as such is not thought to be directly carcinogenic, acetaldehyde, its first metabolite, has been proven genotoxic and mutagenic in the HPRT gene. As mutations in the tumour suppressor gene TP53 are the most common genetic alterations involved in human cancers, especially esophageal tumours, the aim of this work was to establish the mutational pattern induced by acetaldehyde in vitro on the TP53 gene, and to compare this pattern with that found in human alcohol-related tumours. For this purpose, we used a functional assay in yeast, the FASAY (functional analysis of separated alleles in yeast), after in vitro exposure of human normal fibroblasts AG1521 to acetaldehyde. We noted 35 mutations, of which 32 were single-nucleotide substitutions including 2 nonsense and 30 missense mutations. The pattern showed that the main mutations were G>A transitions (n=23, of which 14 in CpG sites), followed by G>T transversions (n=4), A>G transitions (n=2) and A>T transversions (n=2). Other mutations were one-base insertion and two deletions, leading to frameshifts. Eleven mutations (31%) were located in TP53 hot-spots in codons 245, 248, 249 and 273. Finally, we compared this pattern with that found for esophageal cancers in humans. These results support the notion that acetaldehyde plays a role in TP53 mutations in esophageal cancers. The key feature of this approach is that mutagenesis is directly studied in a key gene in human carcinogenesis, allowing direct comparison of mutational patterns with those in human tumours.

Identification of new single nucleotid polymorphisms (SNP) in alcohol dehydrogenase class IV ADH7 gene within a French population.

Many epidemiological studies have explored the possible link between the susceptibility to alcohol related cancers, such as oesophageal cancers, and genetic variants of alcohol dehydrogenases (ADHs). Alcohol dehydrogenase class IV ADH7 is mainly expressed in the upper aero-digestive tract and is involved in the first pass ethanol metabolism. As far as we know, no study has described single nucleotide polymorphisms (SNPs) within ADH7 exons in the Caucasian population. Therefore, in a pilot study, we used the denaturing high performance liquid chromatography (DHPLC) method to screen 49 oesophageal cancer cases for SNPs in the ADH7 gene. A total of 5 SNPs was observed in this study: one SNP in the 5' non-translated regions of exon 1, two SNPs in introns 3 and 4 and two others SNPs in exons 3 and 4. The SNP located in the exon 1, which has never been described before, occurred in a reverse TATA box whereas the SNP of exon 3 was a non-silent polymorphism. Because these two SNPs could potentially affect the transcription and/or the enzyme activity, their distribution was evaluated in a representative sample of healthy Caucasians (n = 89) recruited in Lower Normandy. Frequencies of heterozygous samples ranged from 11% (exon 3) to 28% (exon 1). No homozygous samples were found. In this pilot study, the DHPLC method was suitable for both SNP screening and genotyping and allowed the detection of five SNPs in the ADH7 gene, two of which have never been described before, among the European population.

GSTM1, GSTT1, GSTP1 and CYP1A1 genetic polymorphisms and susceptibility to esophageal cancer in a French population: different pattern of squamous cell carcinoma and adenocarcinoma.

AIM: To evaluate the association between CYP1A1 and GSTs genetic polymorphisms and susceptibility to esophageal squamous cell carcinoma (SCC) and esophageal adenocarcinoma (ADC) in a high risk area of northwest of France. METHODS: A case-control study was conducted to investigate the genetic polymorphisms of these enzymes (CYP1A1*2C and GSTP1 exon 7 Val alleles, GSTM1*2/*2 and GSTT1*2/*2 null genotypes). A total of 79 esophageal cancer cases and 130 controls were recruited. RESULTS: GSTM1*2/*2 and CYP1A1*1A/*2C genotype frequencies were higher among squamous cell carcinomas at a level close to statistical significance (OR = 1.83, 95% CI 0.88-3.83, P = 0.11; OR = 3.03, 95% CI 0.93-9.90, P = 0.07, respectively). For GSTP1 polymorphism, no difference was found between controls and cases, whatever their histological status. Lower frequency of GSTT1 deletion was observed in ADC group compared to controls with a statistically significant difference (OR = 13.31, 95% CI 1.66-106.92, P<0.01). CONCLUSION: In SCC, our results are consistent with the strong association of this kind of tumour with tobacco exposure. In ADC, our results suggest 3 distinct hypotheses: (1) activation of exogenous procarcinogens, such as small halogenated compounds by GSTT1; (2) contribution of GSTT1 to the inflammatory response of esophageal mucosa, which is known to be a strong risk factor for ADC, possibly through leukotriene synthesis; (3) higher sensitivity to the inflammatory process associated with intracellular depletion of glutathione.

Assessment of DHPLC usefulness in the genotyping of GSTP1 exon 5 SNP: comparison to the PCR-RFLP method.

Single-nucleotide polymorphism (SNP) analysis can be performed by several methods such as PCR-RFLP, real time PCR and mass spectrometry. Denaturating High Pressure Liquid Chromatography (DHPLC) analysis allows the detection of DNA mutations in heteroduplex samples. GSTP1 exon 5 gene presents a single-nucleotide polymorphism (a to g) that results into an amino-acid substitution (Ile to Val). Ile and Val variants are identified respectively by a and b alleles. This polymorphism affects enzyme activity and is highly frequent within Caucasian populations and therefore widely studied in the context of SNP related to cancer susceptibility. Our goal was to evaluate DHPLC usefulness in detecting a well-known SNP in comparison to PCR-RFLP, in the field of molecular epidemiological studies. Fifty Caucasian people were genotyped by both methods. Heterozygous samples were identified easily at two temperatures using the DHPLC method. Discrimination between a/a and b/b homozygous genotypes was done by pooling every homozygous sample with a known a/a sample. Our genotyping using both methods resulted in the characterisation of 32 (64%) a/a homozygous, 18 (36%) a/b heterozygous and 5 (10%) b/b homozygous. All samples were also identically genotyped by the two methods. Our results show that DHPLC is a good alternative to classical PCR-RFLP method in genotyping SNPs. Advantages of this chromatographic method were no restriction site needed and a reduced technical time thanks to an automated injection. Moreover, unlike classical RFLP gel analysis, DHPLC chromatograms provided objective criteria for sample classification.

Simultaneous use of DGGE and DHPLC to screen TP53 mutations in cancers of the esophagus and cardia from a European high incidence area (Lower Normandy, France).

We investigated TP53 mutation patterns in cancers of the esophagus and cardia of patients coming from Lower Normandy, a region situated in the highest incidence area in Europe. To screen tumor samples, we first used denaturing gradient gel electrophoresis (DGGE), a well-characterized technique which constituted our reference method. Then the results were compared with those obtained by denaturing high performance liquid chromatography (DHPLC), a recent and automatic screening technology. Analysis of the TP53 mutations profile showed that the detected alterations were mainly point mutations. Ninety-seven percent (33/34) of esophageal squamous cell carcinoma samples presented at least one mutation or polymorphism. The proportion of somatic, non-silent and sequence-confirmed mutations was 76% (26/34). The most common substitutions were G-->A transitions, which could be related to nitrosamines, acetaldehyde or factors prone to producing mucosal irritation, like hot beverages. G-->T transversions, which were also frequently detected, could originate from benzo[a]pyrene in tobacco smoke. A-->T transversions were not revealed in our series, which constitutes a discordance with mutational spectra already performed in north-western France. Concerning adenocarcinoma of the esophagus and cardia, the alteration frequency was 69% (11/16), with a majority of G-->A transitions at CpG dinucleotides. They are probably related to endogenous process mediated by inflammatory diseases like gastro-esophageal reflux and Barrett's esophagus. The main advantage provided by DHPLC was its ease of application. However, the optimization steps turned out to be quite critical, especially for sequences with high melting temperatures embedded in lower melting temperature fragments. Considering only the common sequences analyzed by the two techniques, four of the 46 positive samples detected by DGGE were not revealed by DHPLC. This result stresses the limited sensitivity of DHPLC compared with DGGE under the conditions described in this study.